Worm Breeder's Gazette 11(1): 69

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Nuclear Protein Extracts and Mobility Shifts (or Eight is not Enough)

William K. McCoubrey, Jr. and Philip M. Meneely

At the meeting in May there was considerable interest in the 
protocol we have been using to obtain nuclear extracts.  The procedure 
we use to obtain the nuclei is that described by Dixon et al (WBG 2 (3)
:73-74) except that PMSF is present at 1 mM in all buffers and 1 mM 
DTT is used in place of mercaptoethanol.  The efficiency depends in 
great part on the tightness of the homogenizer used to shear the worms.
The nuclei are washed twice in 10 ml of TKM (50 mM Tris-HCI (7.5), 
25 mM KCL and 5 mM MgCl2) and resuspended in 3 volumes of TKM.  At 
this point the volume of nuclei is sufficiently small (0.05-0.2 ml) to 
carry out the remaining steps in microfuge tubes.  EDTA is added to 0.
01 N and an equal volume of 2X Iysis buffer (1X= 40 mM Tris-HCI (7.5),
1 mM EDTA, 0.5 mM DTT, 1 M NaCl, 10% glycerol) is added.  Tubes are 
mixed gently for 5 minutes in the cold and one-half volume of 18% 
PEG8000 in 1X Iysis buffer is added slowly with gentle mixing.  The 
tubes are slowly rotated in the cold for 1 hour and nucleic acids are 
removed by centrifugation (17000 G for 20 minutes).  This crude 
supernatant appears to be free of nucleases and proteases and can be 
used directly or dialyzed against your favorite buffer using micro 
dialysis with membranes having a 12 kD cut-off, protein concentration 
decreases by a factor of about 2 during dialysis.  Two grams of worms 
yields 0.5 to 1 mg of protein before dialysis.  The extracts have 
little contamination by cytoplasmic components as the vitellogenins 
appear to be absent when SDS-PAGE is performed.
Using extracts from mixed stage and sex N2s, we have repeated the 
gel retardation experiments which we described at the meeting.  
Specific binding can be demonstrated with a double-stranded substrate 
containing the TATTGAAA sequence which we have suggested is an X-
linked binding site used in X/A ratio assessment.  This binding is 
competed by double-stranded oligos which contain the octamer but not 
by those which lack it.  The negatives include a sequence which 
precisely deletes the 8 base pairs but leaves 56 nucleotides around 
the site intact.  Single stranded oligonucleotides, including the two 
complementary strands annealed to give a strong specific competitor do 
not compete for binding.  Curiously, the 8 bp sequence by itself does 
not compete even when ligated into a large concatemer despite the fact 
that it is feminizing in the microinjection assay which we have 
previously described (WBG 10 (1): 62-63).